Intramolecular fluorescence resonance energy transfer and living cell imaging of novel pyridyltriphenylamine dye

Optical Materials 57 (2016) 93e101

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Intramolecular fluorescence resonance energy transfer and living cell imaging of novel pyridyltriphenylamine dye Duojun Cao, Ying Qian* School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu 211189, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 March 2016 Received in revised form 13 April 2016 Accepted 13 April 2016

A novel pyridyltriphenylamine-rhodamine dye PTRh and a pyridyltriphenylamine derivative PTO were synthesized and characterized by 1H NMR and HRMS-MALDI-TOF. PTRh performed typical fluorescence resonance energy transfer (FRET) signal from pyridyltriphenylamine to rhodamine along with notable color change from green to rose when interacting with Hg2þ in EtOH/H2O. And PTRh as a ratiometric probe for Hg2þ based on FRET could achieve a very low detection limit of 32 nM and energy transfer efficiency of 83.7% in aqueous organic system. On the other hand, spectra properties of PTO in its aggregates, THF/H2O mixed solution and silica nanoparticles (Si-NPs) dispersed in water were investigated. And the results indicated PTO exhibited bright green fluorescence in solid state, and PTO was successfully encapsulated in silica matrix (30e40 nm), emitting bright blue fluorescence with 11.7% quantum yield. Additionally, living cell imaging experiments demonstrated that PTRh could effectively response to intracellular Hg2þ and PTO-doped Si-NPs were well uptaken by MCF-7 breast cancer cells. It could be concluded that the chromophores are promising materials used as biosensors. © 2016 Elsevier B.V. All rights reserved.

Keywords: Fluorescence resonance energy transfer Fluorescent silica nanoparticles Living cell imaging Pyridyltriphenylamine

1. Introduction Ratiometric sensors based on fluorescence resonance energy transfer (FRET) have gained extensive attention in the science field of biomedicine and environment because of their salient figure of merits over conventional fluorescence intensity-based probes [1e5]. FRET-based probes can not only circumvent disadvantages of probes based on other mechanisms like intramolecular charge transfer (ICT) but simultaneously and accurately record the ratio of two emission intensities at different wavelengths in the presence and absence of analytes [6,7]. Therefore, a number of probes based on FRET mechanism have been designed to detect and trace special analytes. Of these FRET systems, rhodamine-based FRET system has become an efficient and practical ratiometric chemosensors, especially for heavy metal ions, like Hg2þ, because rhodamine derivatives are characterized with excellent photophysical properties, such as high fluorescence quantum yield, long absorption and emission wavelengths and large molar extinction coefficient and specifically, ring-opened rhodamine spirolactam triggered by specific guests [8e10]. And in rhodamine-based FRET systems,

* Corresponding author. E-mail address: [email protected] (Y. Qian). http://dx.doi.org/10.1016/j.optmat.2016.04.024 0925-3467/© 2016 Elsevier B.V. All rights reserved.

rhodamine fluorophores generally act as energy acceptor. And in order to constitute rhodamine platform with high energy transfer efficiency (ETE) and large degree of overlapping, diverse fluorophores including fluorescien [11e13], pyrene [14e17], BODIPY [18e20], coumarin [21,22] and naphthalimine [23e25] derivatives have been incorporated as energy donor in FRET systems by far. Numerous rhodamine-based FRET probes with high sensitivity and selectivity for Hg2þ detection have been reported recently due to toxic effects of mercury on ecosystems and human health. Qian et al., combinated a BODIPY fluorophore with rhodamine B derivative through a conjugated phenyl-ethynyl-phenyl spacer to detect Hg2þ based on FRET mechanism with 99% ETE. And the ratiometric probe can be practically applied in living cells [26]. Kumar's group developed a FRET chemosensor based on rhodamine and napthalimide for Hg2þ with detection limit of 10 ppb [27]. Wang et al. introduced a reaction-based consisting of a coumarin donor and rhodamine thiosemicarbazide acceptor to detect Hg2þ. In the presence of Hg2þ, the rhodamine moiety was in the ring-opened form by Hg2þ-promoted formation of 1,3,4-oxadiazoles from thiosemicarbazole [28]. Han's team recently attached phenothiazine fluorophore with rhodamine B using a hydrazine hydrate as spacer which allowed ratiometric detection of Hg2þ in acetonitrile [29]. In the FRET process, energy is transferred from the phenothiazine moiety to the rhodamine B moiety. It could be concluded that only

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one kind of rhodamine derivatives as energy acceptor was used in these works and fluorophores as energy donor could be various. On the other hand, rapid development of fluorescent nanoparticles has created new opportunities for biological and medical applications [30,31], such as gene delivery [32,33], bioimaging [34]. Therefore, the researches on multifunctional nanoparticles have attracted extensive attention in the past decades. Of these nanoparticles, dye-doped silica nanoparticles are most vigorously investigated and utilized as biosensors because of their excellent features such as facile surface modification, low-cost, high hydrophilicity and biocompatibility, which breaks a new path for applications of hydrophobic dye [35,36]. To date, much effort have been dedicated to fabricating novel dye-doped silica nanoparticles. Lu's group synthesized a new kind of silica nanoparticles encapsulating Cy-5 by a reverse microemulsion method. These nanoparticles are uniform-sized and monodispersed, and exhibit high fluorescence intensity, photostability, low cytotoxicity and good biocompatibility, which successfully label living cells [37]. Besides, Yan et al. developed fluorescein-doped silica nanoparticles (FSNPs) functionalized with D-arabinose (Ara), which showed strong and specific interactions with M. smegmat is over other bacteria like E. coli ORN 178 and caused the mycobacteria to aggregate. And then Ara-functionalized nanoparticles facilitating mycobacterial aggregation was used to detect M. Smegmat is at a concentration as low as 104 CFU per mL, potentially providing an alternative testing method for the detection and imaging of TB [38]. Inspired by the ratiometric probes based FRET reported, we have designed and synthesized a novel ratiometric fluorescent probe PTRh (Scheme 1) consisting of a pyridyltriphenylamine donor and a rhodamine acceptor, for detection of Hg2þ in EtOH/H2O system. And to the best of our knowledge, there are few articles about tripheylamine-rhodamine system as sensors for cations [39]. However, there has yet been no report of FRET system between pyridyltripheylamine and rhodamine B. And according to our previous works, triphenylamine derivatives exhibit excellent electron donating ability and optoelectronic properties, like two-photon induced fluorescence and aggregation-induced fluorescence [40e43]. In this regard, tripheylamine-based fluorophore was chosen and incorporated into rhodamine acceptor. And vinylpyridine conjugated tripheylamine donor can not only provide high degree of spectra overlap between donor emission and acceptor absorption but increase the water solubility of PTRh to some degree. In this way, the probe PTRh was applied in the ratiometric

detection of Hg2þ and incubated with living cells. Meanwhile, owing to excellent photophysical properties and special stereostructure of triphenylamine derivatives, we have synthesized another triphenylamine-based chromophore, PTO (Scheme 1). And this compound showed bright solid fluorescence under ultraviolet lamp. However, the bioapplications of PTO were limited by its poor solubility and biocompatibility in water. Herein, PTO encapsulating nanoparticles with unique structural characteristics were fabricated and tentatively used as biosensor for cell imaging.

2. Experimental 2.1. Materials and methods 4-vinylpyridine (AR, KangtuoChem, Shanghai, China), palladium acetate, tri-o-tolylphosphine and sodium hydride, Rhodamine B, hydrazine hydrate (AR, Aladdin Industrial Co., Shanghai, China), 3aminopropyltriethoxysilane (APTES, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China), Surfactant Aerosol OT (AOT, Aladdin Industrial Co., Shanghai, China), triethoxyvinylsilane (VTES, Aladdin Industrial Co., Shanghai, China). Several important intermediates were prepared based on previous works, which were [[4-[5-(4-tertbutylphenyl)-1,3,4-oxadiazol-2-yl] phenyl]methyl] triphenylphosphonium bromide [44], 4-[bis(4-iodophenyl)amino] benzaldehyde [45], Rhodamine B hydrazine (RBH) [46,47], and 4-[bis[4-[2-(4pyridinyl)ethenyl]phenyl]amino] benzaldehyde (TP) [41,48]. The organic solvents were purified and dried based on standard protocols. All other chemicals and reagents were used without further purification. All the compounds structures were characterized by 1H NMR (Bruker spectrometer, 300 MHz, tetramethylsilane as the internal standard), and Mass spectrometer (Ultraflex II, MALDI-TOF). UVevisible absorption spectra were determined on a Shimadu UV-3600 spectrophotometer. Fluorescence spectra were measured on an HORIBA FL-4 max spectrometer. The melting points were measured on a Microscopic Melting Point Meter X-4. TEM images were recorded on a JEM-2100EX microscopeoperated at 200 kV. The cell imaging equipment used in our experiment were Upright Fluorescence Microscope (Imager A1) provide by ZEISS, Germany. The solutions of cations were prepared by dissolving the corresponding nitrate (Agþ, Al3þ, Ca2þ, Cd2þ, Co2þ, Cu2þ, Fe3þ, Kþ, Mg2þ, Naþ, Ni2þ, Pb2þ, Zn2þ, Pb2þ) and chloride (Fe2þ) or perchlorate (Hg2þ) salts in deionized water. The stock solution of probe PTRh (104 M) was prepared in ethanol. And the working solutions

Scheme 1. Structures of dye PTRh and PTO.

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Scheme 2. Synthesis routes for target molecules.

were obtained by diluting with PBS (pH ¼ 7.4, 10 mM) buffer solution. And the quantum yields of PTO and PTRh before combining with Hg2þ were determined using quinoline in sulfuric acid (0.1 M) as the standard (FF ¼ 55%) while that of PTRh after interacting with Hg2þ was measured using rhodamine B in EtOH as the standard (FF ¼ 65%). 2.2. Synthesis of PTI In a three-necked flask, [4-[5-(4-tert-buthylphenyl)-1,3,4oxadiazol-2-yl]phenyl triphenylphosphonium bromide (0.65 g, 1.03 mmol) was dissolved in dry THF (20 mL) in ice-water bath under N2. To above solution, NaH (0.2 g, 8.33 mmol) was added and the mixture was stirred for 30 min. And then the solution of 4[bis(4-iodophenyl)amino] benzaldehyde (0.539 g, 1.03 mmol) in dry THF (10 mL) was dropwise added to the orange solution. After that, ice-water bath was removed and the resulting mixture was heated and refluxed for 8 h. The excess solvent was distilled under reduced pressure, and the mixture was poured into a certain amount of methanol. Yellow precipitate was dried under vacuum and purified by silica-gel column chromatography (dichlomethanepetroleum ether). A yield of 40% (0.32 g) yellowish-green solid was

obtained. M.P. 229e231

 C. 1H

NMR (300 MHz, CDCl3, Me4Si),

d (ppm): 8.12 (dd, 4H, J1 ¼ 8.37 Hz, J2 ¼ 8.55 Hz), 7.65 (d, 2H,

J ¼ 8.16 Hz) 7.57 (d, 6H, J ¼ 8.46 Hz), 7.45 (d, 2H, J ¼ 8.34 Hz), 7.20 (d, 1H, J ¼ 16.32 Hz), 7.10 (s, 2H), 7.05 (d, 1H, J ¼ 5.34 Hz), 6.87 (d, 4H, J ¼ 8.46 Hz), 1.41 (s, 9H). Calculated for [MþH]þ: 800.06, Found: 800.40. 2.3. Synthesis of PTO At room temperature, in a 50 mL double-necked flask, PTI (0.50 g, 0.63 mmol), 4-vinylpyridine (0.50 mL, 4.50 mmol) and anhydrous K3PO4 (0.40 g, 1.88 mmol) were dissolved in dry DMF (5 mL) with well stirring under N2 protection for 30 min. Then, Trio-tolylphosphine (0.048 g, 0.16 mmol) was added to the mixture. The solution was stirred for another 30 min under N2 before addition of palladium acetate (0.014 g, 0.0625 mmol). The mixture was maintained at 110  C for 2 days. After the reaction, the resulting mixture was cooled to room temperature and added to certain amount of water. Yellow precipitate was formed, dried under vacuum and purified by silica-gel column chromatography (dichlomethane-ethyl acetate-methanol) to give orange solid, 0.23 g. Yield: 48%. M.P.165e167  C. 1H NMR (300 MHz, CDCl3, Me4Si), d (ppm): 8.57 (d, 4H, J ¼ 5.58), 8.10 (dd, 4H, J1 ¼ 8.25 Hz, J2 ¼ 8.40 Hz), 7.65 (d, 2H, J ¼ 8.31), 7.56 (d, 4H, J ¼ 9.90), 7.47 (d, 6H, J ¼ 8.34), 7.36 (d, 4H, J ¼ 5.67), 7.29 (d, 2H, J ¼ 11.40), 7.19e7.06 (m, 8H), 6.95 (d, 2H, J ¼ 16.26), 1.38 (s, 9H). Calculated for [MþH]þ: 754.3546, Found: 754.3469. 2.4. Synthesis of PTRh

Fig. 1. Absorption and fluorescence spectra of PTRh (20 mM) in EtOH/H2O (6:4, v/v, 10 mM PBS, pH ¼ 7.4), inset: fluorescence picture under UV lamp.

In a 50 mL two-neck flask, rhodamine B hydrazide (0.321 g, 0.703 mmol) was added into ethanolic solution dissolved TP (0.224 g, 0.47 mmol). Then, to the mixture catalyst, acetic acid (0.1 mL), was added and stirred for 10 min under nitrogen. After that, the system was refluxed at 80  C for 8 h. The resulting solution was allowed to cool to RT, yellow solid separated out. The crude product was obtained by filtration and purified by silica-gel column chromatography (dichlomethane-metanol). A yield of 58% orange solid (0.25 g) was gotten. M.P. 180e182  C.1H NMR (300 MHz, CDCl3, Me4Si), d (ppm): 8.74 (s, 1H), 8.55 (d, 4H, J ¼ 4.95 Hz), 7.98 (d, 1H,

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Fig. 3. Fluorescence titration spectra of PTRh (20 mM) in EtOH/H2O (6:4, v/v, 10 mM PBS, pH ¼ 7.4) upon gradual addition of Hg2þ (0e10 equiv.) (a), inset: upon gradual addition of Hg2þ (2e4.5 equiv), linear variation of fluorescence intensity ratio (F585/ F515) and fluorescence pictures of probe in absence and presence of Hg2þ in sunlight and under UV lamp excited at 365 nm (b).

(20 mL) was added and the mixture was stirred for another 24 h at room temperature. After the formation of fluorescent silica nanoparticles, surfactant AOT, cosurfactant 1-butanol, unloaded dye, residual VETS and APTES were removed by dialyzing against deionized water in an 8e14 kDa cut-off cellulose membrane for

Fig. 2. Absorption spectra (a) and absorbance change (b) of PTRh (20 mM) toward 20 equiv. various metal cations in EtOH/H2O (6:4, v/v, 10 Mm PBS, pH ¼ 7.4), and the corresponding color changes in sunlight.

J ¼ 6.30 Hz), 7.46 (d, 4H, J ¼ 7.83 Hz), 7.40 (d, 4H, J ¼ 8.43 Hz), 7.34 (d, 4H, J ¼ 5.37 Hz), 7.27 (s, 1H), 7.22 (s, 1H), 7.11 (d, 1H, J ¼ 7.14 Hz), 7.04 (d, 4H, J ¼ 8.43 Hz), 7.00 (d, 2H, J ¼ 8.37 Hz), 6,90 (d, 2H, J ¼ 16.20 Hz), 6.52 (d, 2H, J ¼ 8.82 Hz), 6.43 (s, 2H), 6.25 (d, 2H, J ¼ 8.79 Hz), 3.33 (dd, 8H, J1 ¼ 6.87 Hz, J2 ¼ 6.93 Hz), 1.15 (t, 12H, J1 ¼ 6.93 Hz, J2 ¼ 6.96 Hz). Calculated for [MþH]þ: 918.4495, Found: 918.4500.

2.5. Fabrication of PTO-doped amine-modified silica nanoparticles The dye-encapsulated luminescent silica nanoparticles were prepared in the nonpolar core of AOT/DMF/water micelles [30,49e51]. Generally, surfactant AOT (0.44 g) and cosurfactant 1butanol (0.8 mL) were dissolved in deionized water (20 mL) with vigorous stirring to form oil-in-water microemulsion system. Until the mixture became clear, DMF (60 mL) solution containing PTO added to the micelles dropwise. After 5 min sonication, VETS was added and the resulting solution was stirred for 6 h. Finally, APTES

Fig. 4. Job plot (a) of PTRh for the absorbance change at a wavelength of 555 nm, the total concentration of probe and Hg2þ was kept at a constant of 50 mM. And the BenesieHildebrand plot of 1/(A-A0) Versus 1/[Hg2þ] (b).

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3.2. Intramolecular fluorescence resonance energy transfer of the pyridyltriphenylamine-rhodamine dye

Fig. 5. Reversibility of Hg2þ to PTRh by Na2S, excited at 404 nm.

72 h. And then, the dialyzed solution was filtered by through 0.45 mm cut-off cellulose membrane filter and stored at 5  C for later experiments. 3. Results and discussion 3.1. Design and synthesis of the pyridyltriphenylamine dye The final novel compounds, PTRh and PTO, shared on a same electron-rich block, pyridyltriphenylamine, and were conjugated by different linkers, C]N bond for PTRh and C]C bond for PTO. In PTRh system, pyridyltriphenylamine served as energy donor while rhodamine B was energy acceptor. And the purpose of introduction of vinylpyridine was to increase the degree of overlap between donor emission and acceptor absorption, and to enhance water solubility of dye to a certain extent. Similarly, pyridyltriphenylamine and 1,3,4-oxdiazole group acted as electrondonating moiety and electron-accepting unit, respectively, in pconjugated PTO system. Firstly, RBH (1.5 equiv.) reacted with TP by Schiff base condensation to prepare PTRh in yield of 58% under the condition of weak acid. And then, PTO was synthesized throughpalladium-catalyzed Heck coupling reaction that connected PTI to 4-vinylpyridine in 48% yield. And their synthesis routes to access two fluorescent molecules were shown in Scheme 2.

Firstly, the absorption and fluorescence spectra of pure PTRh were investigated in EtOH/H2O (PBS buffer, pH ¼ 7.4) solution (Fig. 1). The maximum absorption and emission peaks for PTRh were 404 nm (3max ¼ 1.20  105 M1 cm1) and 515 nm with a Stokes shift of 5335 cm1, which were characteristic peaks of pyridyltriphenylamine unit, indicating that the rhodamine spirocycle of PTRh was in “off” state. Then, the UVevis spectra responses of PTRh to various common ions were tested in EtOH/H2O system. It was clearly found that PTRh performed excellent selectivity for Hg2þ at 555 nm, suggesting that the ring-opening spirolactam results from Hg2þ binding, and no significant variation in the absorption spectra of the probe was observed with other cations (Fig. 2a and b). Besides, from the fluorescent pictures, PTRh can serve as a “naked-eye” probe for Hg2þ with remarkable color change. It is well known that FRET mechanism is dominated by the distance and scope of spectra overlap between two different chromophores serving as energy donor and acceptor [52,53]. Thus, the overlapped degree of spectra and three dimensional (3D) simulation distances between pyridyltriphenylamine and rhodamine were studied in this paper (Fig. S1). It was interesting to find that large degree of spectra overlap and Schiff-base distance (<10 Å) of two chromophores met the conditions of FRET mechanism. From above discussion, free PTRh displayed a strong emission band centred at 515 nm, which was attributed to pyridyltriphenylamine moiety. But, upon gradual addition of Hg2þ, the emission at 515 nm decreased dramatically while emission band centred at 585 nm appeared and gradually increased, demonstrating that FRET signal occurred from pyridyltriphenylamine to rhodamine. More notably, when the concentration of Hg2þ increased from 0 to 10 equiv., fluorescence intensity ratio (F585/ F515) enhanced about 50 folds (from 0.742 to 37.73). There was a good linear relationship in the concentration range from 2 to 4.5 equiv. of PTRh (R2 ¼ 0.9643) and the detection limit was calculated to be 32 nM (S/N ¼ 3) [54] (Fig. 3 inset). Meanwhile, the energy transfer efficiency (ETE) value was 83.7% based on E ¼ 1  FD=FD0, where FD and FD0 are the fluorescence intensities of the donor in the cassette and the fluorescence intensities of free donor [55,56] (Fig. S2). And the quantum yield of probe PTRh in EtOH/H2O (PBS buffer, pH ¼ 7.4) was measured to be 10.2% while in presence of enough amount of Hg2þ, the value of FF increased to

Scheme 3. Possible binding mode of PTRh with Hg2þ ions.

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Fig. 7. The fluorescence spectrum of PTO in solid state: (a) PTO solid powder excited at 400 nm and the solid fluorescence picture excited at 365 nm under UV lamp (inset), (b) the according CIE chromaticity coordinate of PTO. Fig. 6. Overlay fluorescence imaging pictures of MCF-7 breast cancer cells treated with probe PTRh (10 mM) in absence (a) and presence (b) of Hg2þ (2 equiv.), scale bar ¼ 25 mm.

21%. According to the Job plot analysis (Fig. 4a), the inflection point was at ~0.5, indicating that the composition of PTRh complex was 1:1. Further, the absorbance value 1/(A-A0) varied as a function of 1/ [Hg2þ] in a good linear relationship (R2 ¼ 0.9883) based on BenesieHildebrand expression [57,58] (Fig. 4b), confirming that the probe bound with Hg2þ in a 1:1 binding stoichiometry [59,60], and the association constant was calculated to be 4.5  104 M.1 To ensure the non-interference of other metal ions, the competition experiment was carried out. And it could be seen that PTRh may be used as the probe for Hg2þ with high selectivity (Fig. S3). Besides, the Na2S-addition experiments were conducted to wonder whether the binding of PTRh/Hg2þ species is reversible. Since the value Ka[PTRh/Hg2þ] calculated is much smaller than that of Kd[HgS2]2 (1050 M2) [61], the addition of Na2S may release the free PTRh from the PTRh/Hg2þ complex. And Fig. 5 suggested that the color of the solution could return to green from red with FRET signal turning off. Moreover, the sensing behavior of PTRh could be revived by adding enough Hg2þ once again. Therefore, it could be concluded that ring opening of rhodamine spirolactam was attributed to the Hg2þ chelation-induced rather than other possible mechanisms [62] (Scheme 3).

The last but most important was that the probe PTRh could be successfully used for recognition of intracellular Hg2þ. As shown in Fig. 6, when MCF-7 breast cancer cells were incubated with only probe PTRh for 30 min at 37  C, the cells showed a clear green intracellular fluorescence (Fig. 6a), which indicated that the probe was cell permeable. However, after further treatment with Hg2þ, a

Fig. 8. Absorption and fluorescence spectra of PTO in THF solution (10 m M), inset: the fluorescence picture under UV lamp.

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Fig. 9. Fluorescence spectra of PTO in THF-H2O solution (1:4, v/v, 10 m M) and PTO doped Si-NPs in water (a) and TEM image of nanoparticles (b); inset: fluorescence pictures are pure PTO in THF solution and fluorescent Si-NPs in water excited at 365 nm.

partial fluorescence quenching in the green channel and a strong fluorescence in the red channel were observed (Fig. 6b). Besides, the cancer cells still remained viable and no apparent toxicity and side effects were observed when incubated with probe PTRh. Thus, the probe could be considered as a significant and potential biosensor applied in biological tissues. 3.3. Aggregation-induced fluorescence in nano-aggregates and living cell imaging of pyridyltriphenylamine-oxidiazole dye From the Fig. 7a inset, it was obviously observed oxadiazole conjugated pyridyltriphenylamine (PTO) emitted bright green fluorescence in the solid states under UV lamp. And its solid fluorescence spectrum was presented in Fig. 7a with maximum emission peak locating at 537 nm. The CIE chromaticity coordinate of the chromophore was depicted in Fig. 7b and CIE value was calculated to be (0.4104, 0.5503), which was in good agreement with its fluorescent picture in solid state. PTO was readily soluble in common organic solvents like THF, but insoluble in water. In Fig. 8, the spectra properties of PTO in pure THF solution were presented. The characteristic absorption and emission of PTO in THF solution were centred at 408 nm (3max ¼ 9.20  104 M1 cm1) and 501 nm with a Stokes shift of 4550 cm1. And the quantum yield was measured to be 28.0%. Besides, the fluorescence properties of PTO were studied in the aqueous organic mixed solutions to determine whether the pure PTO can further be applied in physiological environment. However, it was a pity that the fluorescence was gradually quenched when more water was added to its THF solution (Fig. S4). Taking THF

Fig. 10. Fluorescence imaging pictures of MCF-7 breast cancer cells cultivated with PTO loaded silica nanoparticles: overlay of light and dark field (a) and blue channel (b), scale bar ¼ 25 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

solution with water fraction of 80% as an example, there was almost no emission (Fig. 9a). Therefore, we have fabricated amino-group-functionalized SiNPs doped with PTO. And Si-NPs coated with amino groups were positively charged that can present electrostatic interaction with negatively charged cell membrane and enhance the cellular uptake [63]. From the fluorescence spectra (Fig. 9a), there was remarkable difference between THF-H2O solution of PTO and its NPs in water. Compared with its aqueous organic solution, Si-NPs doped with PTO showed a significant hypochromatic shift of 28 nm, which may be attributed to the types of solvents or intra-particle environment [64]. The quantum yield of fluorescent Si-NPs of PTO was 11.7%, suggesting bright blue PTO-loaded Si-NPs were promising to be used in biological systems. And interestingly, TEM image showed these fluorescent NPs were spherical in shape and highly monodispersed in water (Fig. 9a inset and Fig. 9b). The average diameters were 30e40 nm suitable for cellular uptake [65]. Additionally, cell imaging experiment indicated that the blue fluorescence was still clearly distinguished when PTO was doped in silica matrix and

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cultivated with living cells (Fig. 10). Thus, it could be concluded that PTO was successfully loaded in silica core, and it is a really good way for the bioapplications of PTO in forms of fluorescent Si-NPs, as needs further exploring as biosensors. 4. Conclusion In summary, the new fluorescent molecules were designed and synthesized sharing on similar building block, pyridyltriphenylamine. The ratiometric probe PTRh exhibited high selective response for Hg2þ in EtOH/H2O system based on FRET mechanism with 83.7% ETE. And the cell imaging indicated that PTRh could be applied to detect Hg2þ in physiological environment. Then, the other compound with oxadiazol group, PTO, was successfully encapsulated in silica nanoparticles to improve their watersolubility and biocompatibility. And the monodispersed dyedoped Si-NPs with diameter of 30e40 nm maintained the inherent properties of pure dye and emitted strong blue fluorescence when incubated with MCF-7 breast cancer cells. Therefore, the conclusion is that dye PTRh and PTO are potential materials used as excellent biosensors in aqueous biosystems. Acknowledgment We thank the financial support from the National Nature Science Foundation of China (Grant numbers 61178057). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.optmat.2016.04.024. References [1] P. Mahato, S. Saha, E. Suresh, R.D. Liddo, P.P. Parnigotto, M.T. Conconi, M.K. Kesharwani, B. Gangulyand, A. Das, Ratiometric Detection of Cr3þ and Hg2þ by a Naphthalimide-Rhodamine Based Fluorescent Probe, Inorg. Chem. 51 (2012) 1769e1777. [2] K.Z. Huang, M.H. Liu, X.B. Wang, D.S. Cao, F. Gao, K. C Zhou, W. Wang, W.B. Zeng, Cascade reaction and FRET-based fluorescent probe for the colorimetric and ratiometric signaling of hydrogen sulfide, Tetrahedron Lett. 56 (2015) 3769e3773. [3] H.T. Zhang, R.C. Liu, Y.T. William, H.W. Xie, H.P. Lei, H.Y. Cheung, H.Y. Sun, A FRET-based Ratiometric Fluorescent Probe For Nitroxyl Detection in Living Cells, ACS Appl. Mater. Interfaces 7 (2015) 5438e5443. [4] J.L. Tang, C.Y. Li, Y.F. Li, C.X. Zou, A ratiometric fluorescent probe with unexpected high selectivity for ATP and its applications in cell imaging, Chem. Commun. 50 (2014) 15411e15414. [5] X.Y. Guan, W.Y. Lin, W.M. Huang, Development of a new rhodamine-based FRET platform and its application as a Cu2þ probe, Org. Biomol. Chem. 12 (2014) 3944e3949. [6] A. Sahana, A. Banerjee, S. Lohar, B. Sarkar, S.K. Mukhopadhyay, D. Das, Rhodamine-Based Fluorescent Probe for Al3þ through Time Dependent PETCHEF-FRET Processes and Its Cell Staining Application, Inorg. Chem. 52 (2013) 3627e3633. [7] N. Kumar, V. Bhalla, M. Kumar, Resonance energy transfer-based fluorescent probes for Hg2þ, Cu2þ and Fe2þ/Fe3þ ions, Analyst 139 (2014) 543e558. [8] G. Sivaraman, V. Sathiyaraja, D. Chellappa, Turn-on fluorogenic and chromogenic detection of Fe (III) and its application in living cell imaging, J. Lumin 145 (2014) 480e485.
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